Matthew J. Giacalone

Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system

The refinement of tightly regulated prokaryotic expression systems that permit functional expression of toxic recombinant proteins is a continually evolving process. Unfortunately, the current best promoter options are either tightly repressed and produce little protein, or produce substantial protein but lack the necessary repression to avoid mutations stimulated by leaky expression in the absence of inducer. In this report, we present three novel prokaryotic expression constructs that are tightly regulated by L-rhamnose and D-glucose. These expression vectors utilize the Escherichia coli rhaT promoter and corresponding regulatory genes to provide titratable, high-level protein yield without compromising clone integrity. Together, these components may enable the stable cloning and functional expression of otherwise toxic proteins.

Immune responses elicited by bacterial minicells capable of simultaneous DNA and protein antigen delivery

Abstract Recent events surrounding emerging infectious diseases, bioterrorism and increasing multidrug antibiotic resistance in bacteria have drastically increased current needs for effective vaccines. Many years of study have shown that live, attenuated pathogens are often more effective at delivering heterologous protein or DNA to induce protective immune responses. However, these vaccine carriers have inherent safety concerns that have limited their development and their use in many patient populations. Studies using nonliving delivery mechanisms have shown that providing both protein antigen and DNA encoding the antigen to an individual induces an improved, more protective immune response but rarely, if ever, are both delivered simultaneously. Here, non-replicating bacterial minicells derived from a commensal E. coli strain are shown to effectively induce antigen-specific immune responses after simultaneous protein and DNA delivery. These data demonstrate the potential use of achromosomal bacterial minicells as a vaccine carrier.

The use of bacterial minicells to transfer plasmid DNA to eukaryotic cells

The delivery of DNA to mammalian cells is of critical importance to the development of genetic vaccines, gene replacement therapies and gene silencing. For these applications, targeting, effective DNA transfer and vector safety are the major roadblocks in furthering development. In this report, we present a novel DNA delivery vehicle that makes use of protoplasted, achromosomal bacterial minicells. Transfer of plasmid DNA as measured by green fluorescent protein expression was found to occur in as high as 25% of cultured Cos‐7 cells when a novel chimeric protein containing the D2–D5 region of invasin was expressed and displayed on the surface of protoplasted minicells. Based on endoplasmic reticulum stress and other responses, protoplasted minicells were non‐toxic to recipient eukaryotic cells as a consequence of the transfection process. Taken together, these results suggest that bacterial minicells may represent a novel and promising gene delivery vehicle.

Preclinical evaluation of VAX-IP, a novel bacterial minicell-based biopharmaceutical for nonmuscle invasive bladder cancer.

The development of new therapies that can prevent recurrence and progression of nonmuscle invasive bladder cancer remains an unmet clinical need. The continued cost of monitoring and treatment of recurrent disease, along with its high prevalence and incidence rate, is a strain on healthcare economics worldwide. The current work describes the characterization and pharmacological evaluation of VAX-IP as a novel bacterial minicell-based biopharmaceutical agent undergoing development for the treatment of nonmuscle invasive bladder cancer and other oncology indications. VAX-IP minicells selectively target two oncology-associated integrin heterodimer subtypes to deliver a unique bacterial cytolysin protein toxin, perfringolysin O, specifically to cancer cells, rapidly killing integrin-expressing murine and human urothelial cell carcinoma cells with a unique tumorlytic mechanism. The in vivo pharmacological evaluation of VAX-IP minicells as a single agent administered intravesically in two clinically relevant variations of a syngeneic orthotopic model of superficial bladder cancer results in a significant survival advantage with 28.6% (P = 0.001) and 16.7% (P = 0.003) of animals surviving after early or late treatment initiation, respectively. The results of these preclinical studies warrant further nonclinical and eventual clinical investigation in underserved nonmuscle invasive bladder cancer patient populations where complete cures are achievable.

Abstract 5703: Efficient targeted delivery of protein toxins using self-manufacturing nanoparticles (minicells) derived from bacteria

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Significant progress has been made with targeted delivery of small molecule cancer therapeutics. The key to clinical success has been the use of extremely potent drugs conjugated to targeting antibodies. There are also classes of very potent and promising therapeutic macromolecules, including protein toxins and RNAi. However, there are several challenges unique to macromolecular payloads that cannot be adequately addressed when using antibodies alone for targeting. These include manufacturing complexity, blood-based catabolism, low payload capacity, poor loading efficiency, and inefficient endosomal escape. Several of these issues have also impeded clinical success with antibody-decorated liposomes. We are addressing these issues by using self-manufacturing nanoparticles, or minicells, derived from E. coli. Minicells are spherical, non-viable ∼400 nm nanoparticles containing all bacterial components, except the chromosome. Minicells bud off the end of rod-shaped bacteria as a result of unequal cell division after induced overexpression of a gene involved in septum formation. We have developed procedures for purification of minicells, including removal of parental bacteria. Our minicell-producing bacterial strains are genetically engineered to prevent growth outside the laboratory. In addition, we have introduced a lipopolysaccharide biosynthesis mutation, which significantly attenuates endotoxin-related inflammatory responses. We have produced minicells with surface expression of the beta1 integrin-targeting protein invasin. The minicells also contain a bacterially-expressed cholesterol-dependent endosomal membrane disrupting protein, such as listeriolysin O (LLO) or perfringolysin O (PFO), and a potent protein toxin, such as the catalytic domain of diphtheria toxin (DT). Invasin binds with high affinity to certain β1-containing integrins, including α5β1, which is overexpressed in tumor vasculature and several solid tumors. Surface expression of invasin on minicells led to binding and endocytosis into human endothelial cells (HUVECs) and tumor cells lines that express α5β1 integrin. Despite significant internalization, invasin-expressing minicells were not toxic to mammalian cells at doses of ≥10,000 minicells per cell. Inclusion of LLO or PFO and DT led to targeted killing of HUVECs and tumor cells (melanoma, colorectal, pancreatic, lung and hepatocellular carcinoma) with IC50s in the range of 50-500 minicells per mammalian cell. In vivo evaluation of minicell toxicity, immunogenicity, tissue distribution and efficacy is in progress. In summary, our minicell-based targeted delivery platform for macromolecular therapeutics reduces manufacturing time and complexity, significantly increases payload capacity, protects payload from blood-based catabolism, and provides a critically enabling endosomal escape mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5703. doi:1538-7445.AM2012-5703

Abstract A05: Immunity, the colonic environment, and colon cancer

Background: Colorectal cancer (CRC) is the second leading cause of cancer-associated deaths in the US. Evidence from several studies suggests that the colonic immune environment can influence the risk of CRC. Bacterial minicells (MC) are spherical, non-viable nano-sized particles derived from genetically-engineered E. coli (Vaxiion Therapeutics, Inc). MC contain all of the components of parental bacteria they are derived from with the exception of the chromosome, making them non-infectious. MC used here have been engineered to display antitumor activity via integrin-specific cytotoxic effects but evidence suggests they also require a working immune system to be optimally effective in mouse models of cancer. For our colon cancer studies, we are using an immune-competent mouse model, FABP-CreXApcfl/+, which has a conditional deletion of exons 11-12 in one Adenomatous Polyposis Coli (Apc) allele in the epithelium of colon and distal ileum, due to the targeted expression of CRE, leading to colonic polyposis. Hypothesis: Since MC have been shown to have anti-cancer activity in multiple mouse models, but even more importantly because evidence suggests they are more effective in animals with a functional immune system, we hypothesize that MC can change the immune environment of the colon and delay/decrease the onset of colon cancer in our mouse model. Methods: We treated 14 week old FABP-CreXApcfl/+ mice with PBS or 1.5 x 109 MC in PBS intra-rectally once a week until 19 weeks of age (6 treatments total). Mice were then left untreated until 26 weeks of age, euthanized, and colons were collected to measure tumor number and size. Colonic tumor load was also compared to 24 week old untreated FABP-CreXApcfl/+ controls. RNA was isolated from tumor adjacent (TA), small tumor (≤4mm) and large tumor (>4 mm) tissue, converted to cDNA, and qPCR studies were performed. Hprt was used as a housekeeping gene control and fold-change target gene expression was reported in comparison to Hprt expression. Results: The mean number of colonic polyps in 26 week old mice that received MC-treatment during the 14th-19th week of age was significantly lower (2.4±1.5) in comparison with mice that received PBS only (9.3±1.4, p=0.03) or 24 week-old untreated controls (8.0±1.9, p=0.02). Moreover, MC-treatment significantly restricted the development of colonic polyps >4mm in size (0.6±0.2/mouse, i.e. 60% of mice had one large tumor each) in comparison to PBS-treated (3.7±0.8/mouse, p=0.03) and 24 week-old untreated (2.0±0.2/mouse, p=0.01) controls. Furthermore, as per qPCR studies, preliminary work suggests that MC promote the expression of TH1 and cytotoxic T cell-associated immune responses (CD8a, Tbet, Ifng, perforin) as well as the TH17 (Il17a) immune gene marker in the TA areas of the colon. We also found a decline in the expression of pro-inflammatory (il23, Il6), immunosuppressive (Il10, Foxp3) and immune cell recruitment (Cx3cl1) immune markers in the large tumors of MC-treated mice. Furthermore, our preliminary data suggest the presence of ‘low’ and ‘high’ responders in MC-treated mice. ‘Low responders’ have at least one large tumor at 26 weeks of age and lower correlation of TH1 and cytotoxic T cell-associated immune responses in comparison to ‘high responders’ which have an absence of large tumors and display multi-fold higher levels of TH1 and cytotoxic T cell-associated immune responses in comparison to PBS-treated controls. Conclusion: MC significantly delay the development of colonic polyps and our preliminary data suggest that the delay/decrease in tumorigenesis could be because of attenuation of pro-tumor inflammation as well as promotion of anti-tumor immunity in the colon. These studies also suggest our mouse model will be appropriate for the study of low and high responder anti-tumor immune phenotypes in mice, shedding light on what may be occurring in human CRC patients. Citation Format: Mohammad W. Khan, Shingo Tsuji, MengXi Tian, Nairika Meshgin, Shea Grenier, Matthew J. Giacalone, Kathleen L. McGuire. Immunity, the colonic environment, and colon cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A05.